Abstract: Since the completion of the Human Genome Project, a major effort in the biomedical sciences has been to understand the function of proteins using perturbation. Functional screening platforms based on RNAi and CRISPR, in particular, have been instrumental in pursuing this goal. Yet despite their wide use and extraordinary strengths, even todays state-of-the-art platforms are still limited in their capabilities. Two prominent limitations include (1) The inability to study the function of genes that are essential for cellular growth, and (2) the slow dynamics of protein depletion that enable adaptation mechanisms that can influence the results. To overcome these limitations, I propose to develop a novel pooled screening platform based on rapid and direct modulation of proteins in human cells at a genome-scale. This platform will be based on multiplexed endogenous tagging of genes with HaloTag, a bioorthogonal protein domain capable of binding a variety of small molecule ligands that can be used to either increase or decrease the stability of the fusion protein. In this proposal I will optimize pooled multiplexed endogenous gene tagging using a unique intron targeting approach which our lab has developed. Genome-scale HaloTag fusion libraries will be used to screen for effects of acute protein degradation and destabilization, identify genes that are prone for transcriptional adaptation, and identify aggregating proteins in an unbiased manner using a different fluorescence ligands.